Therapeutic agent for ectopic pregnancy

ABSTRACT

A novel therapeutic agent for ectopic pregnancy having a therapeutic effect for ectopic pregnancy, especially unruptured ectopic pregnancy, and a novel method of screening a therapeutic agent for ectopic pregnancy are disclosed. The therapeutic agent for ectopic pregnancy contains as an effective ingredient a suppressor of brain-derived neurotrophic factor (BDNF) and/or of brain-derived neurotrophic factor receptor (TrkB). The method of screening a therapeutic agent for ectopic pregnancy includes measuring the kinase activity of TrkB in the presence of a test substance and the kinase activity of TrkB in the absence of the test substance; and selecting the test substance which decreases the kinase activity of TrkB.

TECHNICAL FIELD

The present invention relates to a therapeutic agent for ectopicpregnancy.

BACKGROUND ART

Ectopic pregnancy is a life-threatening condition in the first trimesterof gestation (Non-patent Document 1). Recent advances in serial hormoneassays and transvaginal ultrasonography facilitates the diagnosis andtreatment of ectopic pregnancy before rupture. Early diagnosis andtimely treatment have resulted in a dramatic decline in mortalitybecause of ectopic pregnancy (Non-patent Document 1). Until the mid1980s, treatment for ectopic pregnancy was exclusively surgical. In1982, the present inventors reported treatment of an interstitialectopic pregnancy in a patient with a 15-day course of intramuscularmethotrexate (MTX) (Non-patent Document 2). Subsequently, MTX treatmenthas been accepted as a medical treatment for unruptured ectopicpregnancy. MTX is a folic acid antagonist that interferes with DNAsynthesis and thus highly toxic to rapidly replicating tissues andmalignant cells. However, signs of advanced ectopic pregnancy, such asdetection of embryonic cardiac activity, high human chorionicgonadotropin (hCG) level, and large (>4 cm) size of conceptus arecontraindications to MTX treatment (Non-patent Document 3). Furthermore,gastric distress, nausea, vomiting, stomatitis, canker sore, anddizziness are commonly observed as side effects of MTX treatment(Non-patent Document 3). Thus, development of more potent and safermedical treatment is needed. Human villous trophoblast is composed ofcytotrophoblast and syncytiotrophoblast layers. Cytotrophoblasts displayhighly proliferative and invasive properties in the first trimester ofgestation, whereas syncytiotrophoblasts are differentiated followingfusion of cytotrophoblasts and have little potential for proliferationthroughout pregnancy. Cytotrophoblasts also differentiate into highlyinvasive cells, called extravillous trophoblasts (EVTs), that break outof the chorionic villi, migrate into maternal decidua, and invademyometrium, leading to a remodeling of the utero-placental arteries foradequate supply of maternal blood necessary for fetal growth.Proliferation of trophoblasts in placental villi is observed in thecytotrophoblasts as well as in the EVTs before they migrate out of thevilli (Non-patent Document 4, Non-patent Document 5).

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophinfamily of proteins known to activate the high affinity tyrosine kinase B(TrkB) receptor together with the pan-neurotrophin low-affinityco-receptor p75 (p75NTR) (Non-patent Document 6). Following BDNFbinding, TrkB receptor plays important roles in cell differentiation,proliferation and survival in different cell types (Non-patent Document6, Non-patent Document 7). Although neurotrophins are widely expressedin the central nervous system and are important for neuronaldifferentiation and survival (Non-patent Document 8), they also playimportant roles in nonneuronal tissues (Non-patent Document 9). Thepresent inventors recently found the expression of TrkB and its ligands,BDNF and neurotrophin-4/5 (NT-4/5) in trophectoderm cells of blastocyststage embryos capable of differentiating into invasive trophoblasts, anddemonstrated promotional effects of BDNF on the proliferation andsurvival of trophectoderm cells before implantation (Non-patent Document10). After implantation, the expression of TrkB and its ligands persistsin placental trophoblast cells, and the present inventors demonstratedautocrine/paracrine regulatory roles of the TrkB signaling system introphoblast cell growth and survival during placental development inmice (Non-patent Document 11). In human, the present inventors furthershowed important autocrine roles of the BDNF/TrkB signaling system inmalignant trophoblastic, choriocarcinoma cell growth (Non-patentDocument 12).

PRIOR ART DOCUMENTS Non-Patent Documents

-   Non-patent Document 1: Berg C J, Chang J, Callaghan W M, Whitehead S    J 2003 Pregnancy-related mortality in the United States, 1991-1997.    Obstet Gynecol 101:289-296-   Non-patent Document 2: Tanaka T, Hayashi H, Kutsuzawa T, Fujimoto S,    Ichinoe K 1982 Treatment of interstitial ectopic pregnancy with    methotrexate: report of a successful case. Fertil Steril 37:851-852-   Non-patent Document 3: American Society for Reproductive Medicine    2008 Medical treatment of ectopic pregnancy. Fertil Steril    90:S206-212-   Non-patent Document 4: Pijnenborg R, Bland J M, Robertson W B,    Brosens I 1983 Uteroplacental arterial changes related to    interstitial trophoblast migration in early human pregnancy.    Placenta 4:397-413-   Non-patent Document 5: Aplin J D 1991 Implantation, trophoblast    differentiation and haemochorial placentation: mechanistic evidence    in vivo and in vitro. J Cell Sci 99:681-692-   Non-patent Document 6: Barbacid M 1994 The Trk family of    neurotrophin receptors. J Neurobiol 25:1386-1403-   Non-patent Document 7: Huang E J, Reichardt L F 2003 Trk receptors:    roles in neuronal signal transduction. Annu Rev Biochem 72:609-642-   Non-patent Document 8: Jones K R, Farinas I, Backus C, Reichardt L F    1994 Targeted disruption of the BDNF gene perturbs brain and sensory    neuron development but not motor neuron development. Cell 76:989-999-   Non-patent Document 9: Ip N Y, Stitt T N, Tapley P, Klein R, Glass D    J, Fandl J, Greene L A, Barbacid M, Yancopoulos G D 1993    Similarities and differences in the way neurotrophins interact with    the Trk receptors in neuronal and nonneuronal cells. Neuron    10:137-149-   Non-patent Document 10: Kawamura K, Kawamura N, Fukuda J, Kumagai J,    Hsueh A J, Tanaka T 2007 Regulation of preimplantation embryo    development by brain-derived neurotrophic factor. Dev Biol    311:147-158-   Non-patent Document 11: Kawamura K, Kawamura N, Sato W, Fukuda J,    Kumagai J, Tanaka T 2009 Brain-derived neurotrophic factor promotes    implantation and subsequent placental development by stimulating    trophoblast cell growth and survival. Endocrinology 150:3774-3782-   Non-patent Document 12: Kawamura N, Kawamura K, Manabe M, Tanaka T    2010 Inhibition of Brain-Derived Neurotrophic Factor/Tyrosine Kinase    B Signaling Suppresses Choriocarcinoma Cell Growth. Endocrinology    151:3006-3014-   Non-patent Document 13: Genbacev O, Schubach S A, Miller R K 1992    Villous culture of first trimester human placenta—model to study    extravillous trophoblast (EVT) differentiation. Placenta 13:439-461-   Non-patent Document 14: Tapley P, Lamballe F, Barbacid 1 M 1992    K252a is a selective inhibitor of the tyrosine protein 2 kinase    activity of the trk family of oncogenes and neurotrophin receptors.    Oncogene 7:371-381-   Non-patent Document 15: Ross A H, McKinnon C A, Daou M C, Ratliff K,    Wolf D E 1995 Differential biological effects of K252 kinase    inhibitors are related to membrane solubility but not to    permeability. J Neurochem 65:2748-2756-   Non-patent Document 16: Liu D, Li C, Chen Y, Burnett C, Liu X Y,    Downs S, Collins R D, Hawiger J 2004 Nuclear import of    proinflammatory transcription factors is required for massive liver    apoptosis induced by bacterial lipopolysaccharide. J Biol Chem    279:48434-48442-   Non-patent Document 17: Kawamura K, Fukuda J, Shimizu Y, Kodama H,    Tanaka T 2005 Survivin contributes to the anti-apoptotic activities    of transforming growth factor alpha in mouse blastocysts through    phosphatidylinositol 3′-kinase pathway. Biol Reprod 73:1094-1101-   Non-patent Document 18: Red-Horse K, Rivera J, Schanz A, Zhou Y,    Winn V, Kapidzic M, Maltepe E, Okazaki K, Kochman R, Vo K C, Giudice    L, Erlebacher A, McCune J M, Stoddart C A, Fisher S J 2006    Cytotrophoblast induction of arterial apoptosis and    lymphangiogenesis in an in vivo model of human placentation. J Clin    Invest 116:2643-2652-   Non-patent Document 19: Nakaigawa N, Yao M, Baba M, Kato S, Kishida    T, Hattori K, Nagashima Y, Kubota Y 2006 Inactivation of von    Hippel-Lindau gene induces constitutive phosphorylation of MET    protein in clear cell renal carcinoma. Cancer Res 66:3699-3705-   Non-patent Document 20: Zuckermann F A, Head J R 1986 Isolation and    characterization of trophoblast from murine placenta. Placenta    7:349-364-   Non-patent Document 21: Le Bouteiller P, Solier C, Proll J,    Aguerre-Girr M, Fournel S, Lenfant F 1999 Placental HLA-G protein    expression in vivo:where and what for? Hum Reprod Update 5:223-233-   Non-patent Document 22: Bischof P, Meisser A, Campana A 2000    Paracrine and autocrine regulators of trophoblast invasion—a review.    Placenta 21 Suppl A:S55-60-   Non-patent Document 23: Koide Y, Aoki T, Hreshchyshyn M M 1971    Effects of hormones, methotrexate, and dactinomycin on benign    trophoblast. Am J Obstet Gynecol 109:453-456-   Non-patent Document 24: James J L, Stone P R, Chamley L W 2006 The    regulation of trophoblast differentiation by oxygen in the first    trimester of pregnancy. Hum Reprod Update 12:137-144-   Non-patent Document 25: Caniggia I, Mostachfi H, Winter J, Gassmann    M, Lye S J, Kuliszewski M, Post M 2000 Hypoxia-inducible factor-1    mediates the biological effects of oxygen on human trophoblast    differentiation through TGFbeta(3). J Clin Invest 105:577-587-   Non-patent Document 26: Semenza G L 2003 Targeting HIF-1 for cancer    therapy. Nat Rev Cancer 3:721-732-   Non-patent Document 27: Shi Q, Zhang P, Zhang J, Chen X, Lu H, Tian    Y, Parker T L, Liu Y 2009 Adenovirus-mediated brain-derived    neurotrophic 1 factor expression regulated by hypoxia response    element protects brain from injury of transient middle cerebral    artery occlusion in mice. Neurosci Lett 465:220-225-   Non-patent Document 28: Martens L K, Kirschner K M, Warnecke C,    Scholz H 2007 Hypoxia-inducible factor-1 (HIF-1) is a    transcriptional activator of the TrkB neurotrophin receptor gene. J    Biol Chem 282:14379-14388-   Non-patent Document 29: Handschuh K, Guibourdenche J, Tsatsaris V,    Guesnon M, Laurendeau I, Evain-Brion D, Fournier T 2007 Human    chorionic gonadotropin produced by the invasive trophoblast but not    the villous trophoblast promotes cell invasion and is down-regulated    by peroxisome proliferator-activated receptor-gamma. Endocrinology    148:5011-5019-   Non-patent Document 30: Watson A L, Palmer M E, Burton G 1995 Human    chorionic gonadotrophin release and tissue viability in placental    organ culture. Hum Reprod 10:2159-2164-   Non-patent Document 31: Kar M, Ghosh D, Sengupta J 2007    Histochemical and morphological examination of proliferation and    apoptosis in human first trimester villous trophoblast. Hum Reprod    22:2814-2823-   Non-patent Document 32: Kawamura K, Kawamura N, Mulders S M,    Sollewijn Gelpke M D, Hsueh A J 2005 Ovarian brain-derived    neurotrophic factor (BDNF) promotes the development of oocytes into    preimplantation embryos. Proc Natl Acad Sci USA 102:9206-9211-   Non-patent Document 33: Klein R, Conway D, Parada L F, Barbacid M    1990 The trkB tyrosine protein kinase gene codes for a second    neurogenic receptor that lacks the catalytic kinase domain. Cell    61:647-656-   Non-patent Document 34: Wang T et al., 2008, Identification of    4-aminopyrazolylpyrimidines as potent inhibitors of Trk kinases, J.    Med. Chem, 12; 51(15):4672-84-   Non-patent Document 35: Somaiah N and Simon G R, 2009, Molecular    targeted therapy in non-small cell lung cancer: an overview of    available agents, J. Thorac. Oncol., 4, S 1045-83-   Non-patent Document 36: Michael D. Sadick et al., 1997, Analysis of    Neurotrophin/Receptor Interactions with a gD-Flag-Modified    Quantitative Kinase Receptor Activation (gD.KIRA) Enzyme-Linked    Immunosorbent Assay, Experimental Cell Research, 234, 354-361-   Non-patent Document 37: Anderson R A et al., 2010, Brain-derived    neurotrophic factor is a regulator of human oocyte maturation and    early embryo development, Fertil Steril, 15, 93(5), 1394-406-   Non-patent Document 38: Nanami KAWAMURA et al., Program and Abstract    of Meeting of the Japan Trophoblastic Diseases Society•Japan    Placenta Association, Vol. 28th-18th Page. 38 (2010),    choriocarcinoma cell growth action and its molecular mechanism of    Brain-derived neurotrophic factor (BDNF)/tyrosine kinase B (trkB)    signal-   Non-patent Document 39: Kazuhiro KAWAMURA et al., Program and    Abstract of Meeting of the Japan Trophoblastic Diseases    Society•Japan Placenta Association: Vol. 28th-18th Page. 65 (2010),    Promotion of implantation and placental development by brain-derived    neurotrophic factor (BDNF) and analysis its molecular mechanism-   Non-patent Document 40: Kazuhiro KAWAMURA et al., Journal of Japan    Society for Reproductive Medicine, Vol. 54 No. 4 Page. 324 (2009),    Identification of novel factor controlling implantation and    plancental development and clarification of its molecular mechanism:    brain-derived neurotrophic factor (BDNF)-   Non-patent Document 41: Kazuhiro KAWAMURA et al., Acta Obstettrica    et Gynaecologica Japonica, Vol. 61 No. 10 Page. 1935-1944 (2009),    Influence by central nerve-related physiologically active substance    on oocyte maturation, embryonic development and implantation-   Non-patent Document 42: Kazuhiro KAWAMURA et al., Folia    Endocrinologica Japonica, Vol. 85 No. 2 Page. 657 (2009), Control of    implantation and plancental development by brain-derived    neurotrophic factor (BDNF) and its molecular mechanism

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a novel therapeuticagent for ectopic pregnancy having a therapeutic effect for ectopicpregnancy, especially unruptured ectopic pregnancy. Another object ofthe present invention is to provide a novel method of screening atherapeutic agent for ectopic pregnancy.

Means for Solving the Problems

The present inventors intensively studied to discover that growth ofcytotrophoblast cells can be suppressed by suppressing the action ofBDNF and/or TrkB, and treatment of ectopic pregnancy can be attainedthereby, to complete the present invention.

That is, the present invention provides a therapeutic agent for ectopicpregnancy, comprising as an effective ingredient a suppressor ofbrain-derived neurotrophic factor (BDNF) and/or of brain-derivedneurotrophic factor receptor (TrkB). The present invention also providesa method of screening a therapeutic agent for ectopic pregnancy, themethod comprising measuring the kinase activity of TrkB in the presenceof a test substance and the kinase activity of TrkB in the absence ofthe test substance; and selecting a test substance which decreases thekinase activity of TrkB. The present invention further provides a methodof screening a therapeutic agent for ectopic pregnancy, the methodcomprising the following steps (a) to (d):

-   (a) preparing model animals in which human placental villi are    transplanted to a renal tissue of a mammal other than human;-   (b) administering a test sample to one (or one population) of the    model animals prepared and raising the animal(s), and administering    only the carrier in the test sample to another (or another    population) of the model animals prepared and raisin the animal(s);-   (c) comparing cytotrophoblast cells and extravillous trophoblast    cells in the renal tissue in the model animal(s) to which the test    sample was administered with cytotrophoblast cells and extravillous    trophoblast cells in the renal tissue in the model animal(s) to    which the test sample was not administered; and-   (d) selecting the test sample as a therapeutic agent for ectopic    pregnancy, which test sample decreased cytotrophoblast cells and    extravillous trophoblast cells in the renal tissue in the model    animal(s) to which the test sample was administered.

The present invention still further provides a suppressor ofbrain-derived neurotrophic factor (BDNF) and/or of brain-derivedneurotrophic factor receptor (TrkB) for use in the treatment of ectopicpregnancy.

The present invention still further provides a method of treatingectopic pregnancy, the method comprising administering an effectiveamount of a suppressor of brain-derived neurotrophic factor (BDNF)and/or of brain-derived neurotrophic factor receptor (TrkB) to a patientwith ectopic pregnancy.

Effects of the Invention

By the present invention, a novel therapeutic agent for ectopicpregnancy having an excellent therapeutic effect for ectopic pregnancyand a screening method thereof were provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the temporal and spatial expression of BDNF, NT-4/5, andTrkB in human placental villi of intrauterine and ectopic pregnancy,observed in the Example below. (A) Temporal expression of BDNF, NT-4/5,and TrkB in human placental villi during first trimester of gestation.BDNF and NT-4/5 protein or TrkB transcript levels were quantified usingELISA (BDNF and NT-4/5) or real-time RT-PCR (TrkB), respectively. Levelsof BDNF and NT-4/5 proteins and TrkB mRNA were detected using samplesobtained at different pregnant weeks (n=4-6 donors). Levels of TrkB mRNAwere normalized using transcript levels of β-actin in the same sample.Columns, mean; bars, SE. *, P<0.05 vs. 6 weeks of pregnancy.Immunohistochemical detection of BDNF and TrkB in human placental villiobtained from donors (B) and diseased tissues from a patient withectopic pregnancy (C), both at 8 weeks of pregnancy. In placental villi,BDNF was found in syncytiotrophoblasts (arrows) and extravilloustrophoblasts (EVTs). In contrast, TrkB was found in cytotrophoblasts(arrowheads) and extravillous trophoblasts. Upper and middle panels arespecific staining, whereas lower panels depict sections stained withnonimmune IgG and serve as controls. Insert: higher magnification of theimage indicated in original figure. (Scale bars, 100 μm.).

FIG. 2 shows the effects of in vitro suppression of endogenous TrkBsignaling on human trophoblast differentiation, observed in the Examplebelow. Villous explants at 6-8 weeks of gestation were cultured inmedium alone (control, C), with different doses of TrkB ectodomain (TrkBEC), with K252a, or the plasma membrane nonpermeable K252b under 3% O₂.(A) Morphological changes in villous explants at 48 and 96 h of culture.Representative images were obtained from villous explants treated withor without TrkB ectodomain (10 μg/ml), K252a (1,000 nM) or K252b (1,000nM). Outgrowth of EVTs was found in the distal end of the villous tipsand inhibited following treatment with either TrkB EC or K252a. (Scalebars, 100 μm.). Inhibition of EVT outgrowth (B) and HLA-G transcriptlevels (C) following suppression of endogenous TrkB signaling at 96 h ofculture. EVT outgrowth was quantified based on the proportions of EVToutgrowth. EVT positive was defined as ≧50% anchoring villous tipsshowed cell outgrowth (n=12-13). Transcript levels for HLA-G weredetermined using real-time RT-PCR. Data were expressed as fold decreasesrelative to controls and normalized to 1.0. Columns, mean; bars, SE. *,P<0.05 vs. control group.

FIG. 3 shows the effects of in vitro suppression of endogenous TrkBsignaling on human trophoblast viability. Villous explants at 6-8 weeksof gestation were cultured in medium alone (control, C), with TrkBectodomain (10 μg/ml), K252a (1,000 nM) or K252b (1,000 nM) under 3% O₂for 96 h. (A) Histological characterization of cell proliferation incultured villous explants using H&E staining (upper), andimmunodetection of PCNA (middle) and Ki-67 (lower). H&E staining showsdecreases in number of villous cytotrophoblasts (arrowheads 1), but notof syncytiotrophoblasts (arrows), and partial detachment of trophoblastlayers (arrowheads 2) following either TrkB EC or K252a treatment. BothPCNA and Ki-67 signals (brown) were decreased in remainingcytotrophoblasts (arrowheads 3) following treatment with differentinhibitors. Inserts: higher magnification of selected areas; M:matrigel. (Scale bars, 100 μm.). (B) Decreases in glucose utilization ofvillous explants during culture following treatment with either TrkB ECor K252a. Media were changed at day 2 of culture and samples wereobtained after 48 h of culture (n=4). Glucose concentrations in mediawere quantified using an enzymatic assay. Columns, mean; bars, SE. *,P<0.05 vs. control group.

FIG. 4 shows the effects of in vitro suppression of endogenous TrkBsignaling on human trophoblast survival, observed in the Example below.Villous explants at 6-8 weeks of gestation were cultured in medium alone(control, C), with TrkB ectodomain (10 μg/ml), K252a (1,000 nM) or K252b(1,000 nM) under 3% O₂ for 96 h. (A) Detection of DNA fragmentation incultured villous explants using in situ TUNEL staining. Cellular nucleicacids were stained using propidium iodide (red signals). The numbers ofpositive apoptosis signals (green fluorescence) were increased in thecytotrophoblasts (arrowheads) following TrkB ectodomain or K252atreatment. (Scale bars, 100 μm.). Inserts: higher magnification selectedareas; arrows: syncytiotrophoblasts. (B) Increases in caspase-3/7activities in cultured villous explants following treatment with eitherTrkB EC or K252a. Data were expressed as fold increases relative tocontrols and normalized to 1 (n=4). Columns, mean; bars, SE. *, P<0.05vs. control group.

FIG. 5 shows the xenotransplantation of human villi into SCID mice as anin vivo model of ectopic pregnancy, observed in the Example below.Villous grafts at 7-8 weeks of gestation were surgically placed underthe kidney capsule of SCID mice and maintained for 1-3 weeks beforehistological (A) and biochemical (B) analyses. (A) Histologicalevaluation of human villi growth in the mouse kidneys during 3 weeksafter xenotransplantation. Human trophoblasts were detected bycytokeratin immunohistochemistry. At 1 week after xenotransplantation,human trophoblasts invaded into renal tissue of mouse kidney (arrows)from original transplantation sites marked by villous cores(arrowheads). At 3 weeks, the areas of mouse kidney occupied by humantrophoblasts were expanded and trophoblast invasion was extended todeeper regions of the kidney. (Scale bars, 400 μm.). (B) Changes ofhCG-β levels in tissue homogenates of grafted kidneys during 3 weeks ofxenotransplantation. Tissue hCG-β levels were quantified using RIA(n=6-15). Points, mean; bars, SE. (C) Identification of EVTs byimmunodetection of HLA-G in the kidney at 2 weeks afterxenotransplantation of human villi. HLA-G was found in trophoblastsinvading into kidney (arrowheads), while HLA-G was absent in other celltypes of trophoblasts stained with cytokeratin. (Scale bars, 200 μm.).

FIG. 6 shows the suppression of endogenous TrkB signaling led to in invivo growth inhibition of human trophoblasts in a model of ectopicpregnancy, observed in the Example below. SCID mice at 1 week afterxenotransplantation of human villi (7-8 weeks of gestation) under thekidney capsule were treated without (vehicle) or with K252a (500 μg/kg),K252b (500 μg/kg), or MTX (1 mg/kg) daily for 7 days. (A-C) Histologicalcharacterization of trophoblast cell proliferation and apoptosis intransplanted villi. Representative images were obtained from resectedkidneys at 8 days after treatment. Cytokeratin (A, upper panels), HLA-G(A, lower panels), and H&E staining (B, upper panels), showed decreasednumbers of invading EVTs and cytotrophoblasts following K252a treatment.(Scale bars, A: 400 μm; B: 100 μm.). Cell proliferation was detectedusing PCNA (B, middle panels) and Ki-67 (B, lower panels)immunostaining, whereas apoptosis was estimated using in situ TUNELstaining (C). PCNA and Ki-67 signals (brown) decreased, whereas TUNELstained nuclei (green fluorescence) increased in cytotrophoblastsfollowing K252a treatment. (Scale bars, 100 μm.). (D-F) Decreased HLA-Gtranscript levels (D) and hCG-β protein levels (E) as well as increasedcaspase-3/7 activities (F) found in renal homogenates with transplantedvilli following treatment with K252a. Samples were obtained from themice at 8 days after treatment (n=10-15). Transcript levels of HLA-G andcaspase-3/7 activities were expressed as fold increases relative tocontrols (vehicle alone) and normalized to 1. Columns, mean; bars, SE.*, P<0.05 vs. control group.

FIG. 7 shows the lack of cell proliferation activity in the migratingEVTs of villous explants, observed in the Example below. Representativeimages were obtained from villous explants at 6-8 weeks of gestationcultured under 3% O₂. Cell proliferation activity in migrating EVTs wasdetermined by immunodetection of Ki-67 and HLA-G, and H&E staining. EVTswere negative for Ki-67, a marker for cell proliferation, but positivefor HLA-G, a specific marker for EVTs (arrows).

FIG. 8 shows the expression of BDNF, and TrkB in human placental villiand mouse renal tissues, observed in the Example below. BDNF and TrkBtranscript levels were quantified using real-time RT-PCR. Levels of BDNFand TrkB mRNA were detected using human villous samples obtained at 8weeks of gestation or mouse renal samples dissected from the kidney(n=4-6 donors or 4 animals). Levels of TrkB mRNA were normalized usingtranscript levels of β-actin in the same sample. Columns, mean; bars,SE. N.D.: not detected.

FIG. 9 shows the expression of Trk ligands (NGF and NT-3) and receptors(TrkA and TrkC), and truncated TrkB in human placental villi duringfirst trimester of gestation, observed in the Example below. Expressionof Trk ligands and receptors mRNAs in the placental villi was detectedby RT-PCR. Levels of β-actin serve as loading controls. No template DNAwas included for negative controls (NC).

MODE FOR CARRYING OUT THE INVENTION

As described above, the therapeutic agent for ectopic pregnancy of thepresent 2 0 invention contains as an effective ingredient(s) asuppressor of BDNF and/or TrkB. The term “suppressor of BDNF and/orTrkB” herein means (1) a substance which suppresses the physiologicalaction of at least one of BDNF and TrkB; (2) a substance whichsuppresses the biding between BDNF and TrkB, or (3) a substance whichsuppresses the production in a cell of at least one of BDNF and TrkB.Examples of the substance (1) include tyrosine kinase inhibitors.Examples of the substance (2) include (i) free TrkB and TrkB fragmentshaving an ability to bind to BDNF, and (ii) antibodies to TrkB or BDNF.Examples of the substance (3) include (i) interfering RNAs against BDNFgene or TrkB gene, and vectors producing such interfering RNAs in acell, and (ii) antisense nucleic acids against BDNF gene or TrkB geneand recombinant vectors producing such antisense nucleic acids in acell. These are now hereinbelow described.

TrkB has a tyrosine kinase activity. As concretely shown in the Examplesbelow, growth of cytotrophoblast cells can be suppressed by inhibitingthe tyrosine kinase activity so that the therapeutic effect againstectopic pregnancy is exerted. Therefore, a tyrosine kinase inhibitor(suppressor) can be used as the effective ingredient of the therapeuticagent for ectopic pregnancy of the present invention. Various tyrosinekinase inhibitors are known and not a few of them are commerciallyavailable. Commercially available tyrosine kinase inhibitors canpreferably be employed. Examples of the known tyrosine kinase inhibitorsinclude, but are not limited to, K252a, AZ-23 (Wang et al. J Med Chem2008, 51, 4672-84; Non-patent Document 34), CEP-701 (Cephalon Inc., WestChester, Pa.), CEP-751 (Kyowa Hakko Kogyo, Tokyo, Japan), CEP-2563(Cephalon Inc.) and CEP-7801 (Somaiah et al. J Thorac Oncol, 2009,4,S1045-83; Non-patent Document 35).

As the tyrosine kinase inhibitor, the compounds represented by Formula(1) or (2) below whose tyrosine kinase inhibitory activities have beendemonstrated in JP 3,344,586 (Patent Document 1) can also be used.

-   (wherein-   a) both of Z¹ and Z² are hydrogen;-   1) R is selected from the group consisting of OH, C₁-C₆ O-n-alkyl    and C₂-C₆ O-acyl;-   2) X is selected from the following group consisting of:-   H;-   CONHC₆H₅ with the proviso that in this case, R¹ and R² are not    simultaneously Br;-   CH₂ Y wherein Y is OR⁷ (wherein R⁷ is H or C₂-C₅ acyl);-   SOR⁸ wherein R⁸ is C₁-C₃ alkyl, aryl or a nitrogen-containing    heterocyclic group;-   NR⁹R¹⁰ wherein R⁹ and R¹⁰ are independently H or C₁-C₃ alkyl, Pro,    Ser, Gly, Lys or C₂-C₅ acyl with the proviso that only one of R⁹ and    R¹⁰ is Pro, Ser, Gly, Lys or acyl;-   SR¹⁶ wherein R¹⁶ is aryl, C₁-C₃ alkyl or a nitrogen-containing    heterocyclic group;-   N₃;-   CO₂CH₃;-   S-Glc;-   CONR¹¹R¹² wherein R¹¹ and R¹² are independently H, C₁-C₆ alkyl, C₆H₅    or C₁-C₆ hydroxyalkyl, or R¹¹ and R¹² together form —CH₂CH₂OCH₂CH₂—;-   CH═NNHCONH₂;-   CONHOH;-   CH═NOH;-   CH═NNHC(═NH)NH₂;

-   CH═NN(R¹⁷)₂ wherein R¹⁷ is aryl;-   CH₂NHCONHR¹⁸ wherein R¹⁸ is lower alkyl or aryl; or-   X and R together form —CH₂NHCO₂—, CH₂OH(CH₃)₂O—, ═O or    —CH₂N(CH₃)CO₂;-   3) R¹¹, R², R⁵ and R⁶ are independently H, or two or less of these    are F, Cl, Br, I, NO₂, CN, OH, NHCONHR¹³, CH₂OR¹³, C₁-C₃ alkyl,    CH₂OCONHR¹⁴ or NHCO₂R¹⁴, wherein R¹⁴ is lower alkyl; CH(SC₆H₅)₂ or    CH(—SCH₂CH₂S—);-   R¹ is CH₂S(O)_(p)R²¹ and R², R⁵ and R⁶ are H wherein p is 0 or 1,    R²¹ is aryl, C₁-C₃ alkyl, or a nitrogen-containing heterocyclic    group,

-   or CH₂CH₂N(CH₃)₂;-   R¹ is CH═NHR²²R²³ and R², R⁵ and R⁶ are H, wherein R²² and R²³ are    independently H, C₁-C₃ alkyl, C(═NH)NH₂ or a nitrogen-containing    heterocyclic group, or R²² and R²³ together form —(CH₂)₄—,    —(CH₂CH₂OCH₂CH₂)—or —CH₂CH₂N(CH₃)CH₂CH₂—, with the proviso that R²²    and R²³ cannot be simultaneously H, and that at least one of R²² and    R²³ are H except for the cases where both of these are alkyl;-   (b) in cases where Z¹ and Z² together represent O, X is CO₂CH₃, R is    OH, and each of R¹, R², R⁵ and R⁶ represents hydrogen).-   The term “lower” herein means C₁-C₆.

The tyrosine kinase inhibitor represented by Formula (2) is shown below.

-   (wherein-   R³ and R⁴ are independently selected from the group consisting of H,    C₁-C₆ alkyl, C₁-C₃ hydroxyalkyl and C₃-C₆ alkenyl, with the proviso    that R³ and R⁴ are not simultaneously H;-   1) both of Z¹ and Z² are hydrogen,-   R¹, R², R⁵ and R⁶ are independently H, or two or less of these are    F, Cl, Br, I, NO₂, CN, OH, NHCONHR¹³, wherein R¹³ is C₆H₅ or C₁-C₃    alkyl, with the proviso that only one of R¹, R², R⁵ and R⁶ is    NHCONHR¹³; CH₂OR¹³; C₁-C₃ alkyl;-   CH₂OCONHC₂H₅; or NHCO₂CH₃;-   2) in cases where Z¹ and Z² together represent O, each of R¹, R², R⁵    and R⁶ is hydrogen).

Among these compounds, K252a employed in the Examples below is asubstance produced by a soil fungus and is widely used as a tyrosinekinase inhibitor. Since this compound is commercially available, thecommercially available product can be conveniently used.

When a tyrosine kinase inhibitor is used as the therapeutic agent forectopic pregnancy of the present invention, the administration route maybe oral route or a parenteral route. In case of a parenteral route, itcan be administered via various usual administration routes such asdirect administration to the site of the ectopic pregnancy, intravenous,intramuscular, subcutaneous, intracutaneous, percutaneous, rectal andinstillation routes. The dose of administration is appropriatelyselected depending on the type of the tyrosine kinase inhibitor, stateof the patient and so on, the dose per adult per day is usually about 1mg to 100,000 mg, preferably 1 mg to 1000 mg. However, needless to say,the dose is not restricted to this range.

In cases where a tyrosine kinase inhibitor is used as the effectiveingredient of the therapeutic agent for ectopic pregnancy of the presentinvention, the therapeutic agent for ectopic pregnancy of the presentinvention may consist of the tyrosine kinase inhibitor or may beformulated into the forms suited for various administration forms usinga pharmaceutically acceptable carrier(s) and/or diluent(s). Methods offormulation and various carriers therefor are well-known in the art offormulation of pharmaceuticals. The pharmaceutically acceptable carrieror diluent may be, for example, a buffer such as physiological saline ora vehicle (sucrose, lactose, corn starch, calcium phosphate, sorbitol,glycine or the like), and a binder (such as syrup, gelatin, gum arabic,sorbitol, polyvinyl chloride, tragacanth or the like), a lubricant(magnesium stearate, polyethylene glycol, talc, silica or the like)and/or the like may be appropriately admixed. Examples of theadministration forms include oral formulations such as tablets,capsules, granules, powders and syrups; and parenteral formulations suchas inhalants, injection solutions, suppositories and liquids. These canbe prepared by generally known formulation methods.

As described above, a substance which suppresses the binding betweenBDNF and TrkB can also be employed as the effective ingredient of thetherapeutic agent for ectopic pregnancy of the present invention.Examples of such a substance include free TrkB and TrkB fragments havingan ability to bind to BDNF. Since free TrkB binds to BDNF, when freeTrkB is administered, the administered TrkB competes with the originalTrkB on the cell membranes and binds to BDNF. As a result, the amount ofBDNF which binds to the original TrkB on the cell membranes isdecreased. Thus, the free TrkB competingly suppresses the bindingbetween the TrkB of the cells and BDNF. The site at which the TrkB onthe cell membranes binds to BDNF is the ectodomain of TrkB. Therefore,as will be concretely described in the Examples below, the ectodomain ofTrkB and TrkB fragments containing the ectodomain also competinglysuppress the binding between BDNF and TrkB similar to the full lengthTrkB, so that they can be used as the effective ingredient of thetherapeutic agent for ectopic pregnancy of the present invention. Thebase sequence of the cDNA of human TrkB gene is shown in SEQ ID NO:1together with the amino acid sequence encoded thereby, and the aminoacid sequence alone extracted therefrom is shown in SEQ ID NO:2. ThecDNA of human TrkB gene and the amino acid sequenced encoded thereby areknown and registered as GenBank Accession No. NM_(—)006180. In the aminoacid sequence of SEQ ID NO:2 (i.e., the amino acid sequence of the fulllength of TrkB), the ectodomain is from −31st amino acid from theN-terminal (hereinafter referred to as “−31aa”) to 397aa. The TrkBfragment composed of this ectodomain can also be used as the effectiveingredient of the therapeutic agent for ectopic pregnancy of the presentinvention. In general, since smaller size of the polypeptide giveseasier preparation and easier intake into the cells, the above-describedTrkB fragment is preferred from these points of view.

In general, it is well-known in the art that there are cases wherein thephysiological activity of a physiologically active protein is retainedeven if the amino acid sequence of the protein is modified such that asmall number of amino acids are substituted, deleted, and/or inserted.Therefore, in addition to the above-described TrkB or the fragmentsthereof, a polypeptide having an amino acid sequence with a sequenceidentity of not less than 90%, preferably not less than 95%, still morepreferably not less than 99% to the amino acid sequence from −31aa to397aa of the ectodomain in the amino acid sequence of SEQ ID NO:2, whichpolypeptide binds to BDNF and exerts a therapeutic effect againstectopic pregnancy can also be used as the effective ingredient of thetherapeutic agent for ectopic pregnancy of the present invention similarto the free TrkB or the ectodomain fragment thereof The sequenceidentity of the amino acid sequence herein means a value calculated byaligning two amino acid sequences such that the number of matched aminoacids is maximum (by insertion of a gap(s), as required) and dividingthe number of matched amino acids by the number of amino acids of thefull-length sequence (in cases where the total number of amino acids isdifferent between the two amino acids, the number of amino acids of thelonger sequence). Such calculation of the homology can be easily carriedout using well-known software such as BLAST. A polypeptide having thesame amino acid sequence as the amino acid sequence of SEQ ID NO:2 orthe same amino acid sequence as the amino acid sequence of −31aa to397aa in this amino acid sequence, except that one to several aminoacids are substituted and/or deleted, and/or one to several amino acidsare inserted and/or added, which polypeptide has an ability to bind toBDNF, and in turn, has a therapeutic effect for ectopic pregnancy, canalso be used as the effective ingredient of the therapeutic agent forectopic pregnancy of the present invention. The 20 kinds of amino acidsconstituting naturally occurring proteins can be classified based on thesimilarity of properties into neutral amino acids having a low-polarside chain (Gly, Ile, Val, Leu, Ala, Met, Pro); neutral amino acidshaving a hydrophilic side chain (Asn, Gln, Thr, Ser, Tyr, Cys), acidicamino acids (Asp, Glu), basic amino acids (Arg, Lys, His) and aromaticamino acids (Phe, Tyr, Trp), and it is known that substitution of aminoacids within each of these classes does not alter the properties of thepolypeptide in most cases. Therefore, when substituting an amino acid(s)in the polypeptide having the amino acid sequence of SEQ ID NO:2 or theectodomain thereof, by substituting the amino acid(s) within each ofthese classes, the probability that the ability to bind to BDNF of thepolypeptide is retained is high.

As is apparent from the fact that there are cases where a fusedpolypeptide constituted by ligating two types of polypeptides eachhaving a physiological activity retains the physiological activities ofthe respective polypeptides, it is well-known by those skilled in theart that there are cases where a polypeptide containing an entirepolypeptide having a physiological activity and an amino acidsequence(s) attached to one or two terminals thereof retains thephysiological activity. Therefore, a polypeptide containing apolypeptide having an ability to bind to BDNF, which former polypeptidehas an ability to bind to BDNF can also be used as the effectiveingredient of the therapeutic agent for ectopic pregnancy of the presentinvention. In this case, although the number of the amino acids attachedto one or both terminals of the above-described polypeptide having anability to bind to BDNF is not limited as long as the resultingpolypeptide has an ability to bind to BDNF, and in turn, having thetherapeutic effect against ectopic pregnancy, in view of the ease ofsynthesis and of the activity per a unit weight, the number of the aminoacids attached to one or both terminals of the polypeptide is preferablyone to several.

In general, polypeptide formulations are widely used in which apolyethylene glycol (PEG) chain or the like is attached to one terminalof the polypeptide in order to make it difficult to be decomposed byproteases in the body. In the therapeutic agent for ectopic pregnancy ofthe present invention too, a polypeptide containing the entirepolypeptide described above and a stabilizing structure such as a PEGchain attached to one terminal thereof can also be used as the effectiveingredient. In cases where the peptide is stabilized by pegylation, thesize in terms of molecular weight of the PEG is several thousands to50,000, preferably about 10,000 to 50,000. The method of binding PEG toone end of a polypeptide is well-known.

In the present specification and claims, the term “modification” of thefree TrkB or of the fragment thereof having the therapeutic effectagainst ectopic pregnancy herein means the above-described polypeptideshaving an amino acid sequence different from the amino acid sequence ofSEQ ID NO:2 or from the amino acid sequence of the ectodomain thereof,and having the ability to bind to BDNF and in turn, having thetherapeutic effect against ectopic pregnancy, as well as thesepolypeptides to which a stabilizing structure such as PEG chain isattached.

In cases where the above-described free TrkB, an ectodomain fragmentthereof or the above-described modification (hereinafter referred to as“BDNF-binding TrkB fragment or the like” for convenience) is used as theeffective ingredient of the therapeutic agent for ectopic pregnancy, theadministration route may be oral route or a parenteral route. In case ofa parenteral route, it can be administered via various usualadministration routes such as direct administration to the site of theectopic pregnancy, intravenous, intramuscular, subcutaneous,intracutaneous, percutaneous, rectal and instillation routes. In view ofthe absorption into the body and of avoiding the decomposition bydigestive enzymes, parenteral administration is preferred. The dose ofadministration is appropriately selected depending on the type of thetyrosine kinase inhibitor, state of the patient and so on, the dose peradult per day is usually about 1 mg to 100,000 mg, preferably 1 mg to1000 mg. However, needless to say, the dose is not restricted to thisrange. In cases where the BDNF-binding TrkB fragment or the like is usedas the effective ingredient too, the formulation can be attained by aconventional method similarly as described above.

Although the BDNF-binding TrkB fragment or the like by itself can beused as the effective ingredient, a recombinant vector containing anucleic acid encoding the BDNF-binding TrkB fragment or the like, whichcan express the BDNF-binding TrkB fragment or the like in a cell canalso be used as the effective ingredient. Various vectors for genetherapy of mammals are known, and not a few of them are commerciallyavailable. Thus, a recombinant vector obtained by inserting a DNAencoding the BDNF-binding TrkB fragment or the like into the cloningsite of a commercially available vector for gene therapy can preferablybe employed. Fee-charging services for inserting a desired gene into avector to construct a recombinant vector for gene therapy are available,and such a fee-charging service may also be used.

Administration itself of the recombinant vector to a mammal can becarried out by a well-known method. That is, preferably, the recombinantvector may be administered parenterally to the tissue in the vicinity ofthe site of ectopic pregnancy to be treated by injection or the like. Asuspension obtained by suspending the recombinant vector in a buffersuch as phosphate buffered saline (PBS) may be administered. Tofacilitate the introduction of the gene vaccine into the cells, anelectric field pulse may be applied to the site of injection. In thiscase, the strength of the electric field is not restricted and usuallyabout 10 V/cm to 60 V/cm, preferably about 25 V/cm to 35 V/cm, and theperiod of keeping the pulse is usually 20 milli seconds to 100 milliseconds, preferably about 40 milli seconds to 60 milli seconds. Thepulse may be usually applied once to 6 times, preferably about twice to4 times. Although the dose of the recombinant vector may beappropriately selected depending on the symptom and the state of thedamaged site of the nerve, the dose is usually about 1 ng to 10 mg,especially about 100 ng to 1 mg in terms of the weight of therecombinant vector.

As a substance which suppresses the binding between BDNF and TrkB, anantibody to BDNF or an antibody to the BDNF-binding TrkB fragment or thelike may also be used. Since BDNF and the BDNF-binding TrkB fragment orthe like are readily available, antibodies to these can be obtained by aconventional method comprising administering BDNF or TrkB as animmunogen to an animal (excluding human) to induce an antibody. Theantibody may be either a polyclonal antibody or a monoclonal antibody,and the monoclonal antibody can also be prepared by the conventionalhybridoma method. In case of a monoclonal antibody, since it isnecessary that the antibody can suppress the binding between BDNF andTrkB, a monoclonal antibody which suppresses the binding between BDNFand TrkB is screened. In case of a polyclonal antibody, since variousantibodies to all of the epitopes in the immunogen are contained, anantibody which suppresses the binding between BDNF and TrkB is obtainedeven without carrying out such a screening.

In cases where the above-described antibody is used as the effectiveingredient of the therapeutic agent for ectopic pregnancy, theadministration route may be oral route or a parenteral route. In case ofa parenteral route, it can be administered via various usualadministration routes such as direct administration to the site of theectopic pregnancy, intravenous, intramuscular, subcutaneous,intracutaneous, percutaneous, rectal and instillation routes. In view ofthe absorption into the body and of avoiding the decomposition bydigestive enzymes, parenteral administration is preferred. The dose ofadministration is appropriately selected depending on the titer of theantibody, state of the patient and so on, the dose per adult per day isusually about 1 mg to 100,000 mg, preferably 1 mg to 1000 mg. However,needless to say, the dose is not restricted to this range. In caseswhere the antibody is used as the effective ingredient too, theformulation can be attained by a conventional method similarly asdescribed above.

A substance which suppresses the production of BDNF or TrkB in the bodymay also be used as the effective ingredient of the therapeutic agentfor ectopic pregnancy of the present invention. Examples of such asubstance include interfering RNAs (iRNAs) against the BDNF gene or TrkBgene. Examples of the substance which suppresses the expression of theBDNF gene or TrkB gene include iRNAs, preferably siRNAs targeting themRNA of the BDNF gene or TrkB gene. An iRNA is a double-stranded RNAcontaining a strand complementary to the target mRNA, which binds to thetarget mRNA and cleave it. An siRNA is a small iRNA having a size ofabout 21 to 23 bases. Since siRNAs have a small size, the synthesisthereof is easy and the cleavage site thereby in the mRNA can easily beselected, using an siRNA is preferred. The technique of suppressing thegene expression by an siRNA is well-known and a number of vendorsproviding the service to design an siRNA targeting the sequence of themRNA (cDNA sequence) presented by a client and to construct arecombinant vector in which the siRNA is incorporated. As describedabove, since the sequence of the cDNA of the TrkB gene is set forth inSEQ ID NO:1, and the base sequence (GenBank Accession No. NM_(—)170735)of the cDNA of the BDNF gene is set forth in SEQ ID NO:3, siRNAs tothese can be easily designed by those skilled in the art. Briefly, ansiRNA is a double-stranded RNA containing a strand complementary to themRNA to be targeted, whose size is usually 21 to 23 bases, and usuallyhas hung over regions at both ends of the double-stranded RNA. The sizeof the hung over regions is 1 to 2 bases, respectively, and the hungover regions may be composed of deoxynucleotide(s). Although thecomplementarity to the mRNA is preferably complete, there are many caseswhere the siRNA has a sufficient cleaving activity even if there is amismatch of about 1 or 2 bases. The hung over regions may not becomplementary. In many cases, it is preferred to design the siRNA suchthat it has a sequence of aa in the sequence of mRNA and subsequent 19to 21 bases, and one having a gc content of about 50% (usually about 45to 55%) is preferred. Further, in order not to be cleaved during thematuration to a mature protein, in many cases, the siRNA is designed tothe site apart from the 5′-end by 50 bases or more.

Although the siRNA may be administered as it is, a recombinant vectorobtained by incorporating a DNA expressing the siRNA into an expressionvector for mammalian cells may also be administered to produce the siRNAin the cells to suppress the expression of the BDNF gene or TrkB gene.Various expression vectors for mammalian cells are commerciallyavailable, and the above-described DNA may be inserted into themulticloning site thereof. Services by vendors who construct theexpression vectors incorporating a DNA expressing an siRNA may also beused.

Although the dose of administration is appropriately selected dependingon the stage of the development of ectopic pregnancy, state of thepatient and so on, in cases where the suppressor is an siRNA, the doseper adult (body weight: 60 kg) per day is usually about 0.01 mg/kg to10mg/kg, especially about 0.1 mg/kg to 5 mg/kg, and in cases where thesuppressor is a recombinant vector expressing the siRNA, the dosethroughout the therapy per adult per day is about 0.01 mg/kg to 10mg/kg, especially about 0.1 mg/kg to 5 mg/kg. However, needless to say,the dose is not restricted to this range.

Further, as the effective ingredient of the therapeutic agent forectopic pregnancy of the present invention, antisense RNAs against theBDNF gene or TrkB gene may also be employed. An antisense RNA has a basesequence complementary to the full length or a part of the mRNA of thetarget gene, and hybridizes with the mRNA to suppress the translation ofthe mRNA and in turn, to suppress the production of the gene product ofthe target gene. Since the base sequences of the cDNAs of TrkB gene andBDNF gene are set forth in SEQ ID NO:1 and SEQ ID NO:3, respectively,antisense RNAs to these genes can also be easily prepared. The size ofthe antisense RNA is not restricted as long as it can specificallyhybridize with the mRNA of the target gene to suppress the translationof the mRNA, the size is usually about 20 bases to the full length ofthe coding region of the mRNA.

Similar to the case of iRNA, although the antisense RNA itself may alsobe administered, a recombinant vector obtained by incorporating a DNAexpressing the antisense RNA into an expression vector for mammaliancells may be administered to produce the antisense RNA in the cells tosuppress the expression of the BDNF gene or TrkB gene. Variousexpression vectors for mammalian cells are commercially available, andthe above-described DNA may be inserted into the multicloning sitethereof.

The dose of administration of the antisense RNA is appropriatelyselected based on the stage of the development of the ectopic pregnancy,the state of the patient and so on, and the dose may be about the sameas the above-described dose of iRNA.

Based on the discovery that the suppression of the signal of BDNF/TrkBsuppresses the growth of the cytotrophoblast cells and the extravilloustrophoblast cells differentiated from the cytotrophoblast cells, thepresent invention also provides the following screening method:

That is, the present invention provides a method of screening atherapeutic agent for ectopic pregnancy, the method comprising measuringthe kinase activity of TrkB in the presence of a test substance and thekinase activity of TrkB in the absence of the test substance; andselecting the test substance which decreases the kinase activity ofTrkB.

Here, as the test sample, low molecular compounds, peptides, nucleicacid molecules, antibodies and the like may be employed. The kinaseactivity of TrkB can be measured by, for example, detecting theautophosphorylation of TrkB using an anti-phosphotyrosine antibody,although the method is not restricted thereto. Here, the kinase activityof TrkB in the cells is preferably measured.

To effectively measure the kinase activity of TrkB, various devisingsmay be employed. For example, the method such as that described in M. D.Sadick et al., 1997, Exp. Cell. Res., 234, 354-361 (Non-patent Document36) may be employed. That is, TrkB fused with a peptide with 26 aminoacid residues of glycoprotein D is expressed in CHO cells, and BDNF isextracellularly administered thereto to activate TrkB. The cells arethen lysed and TrkB is captured on the well coated with an antibodyspecific to the peptide of glycoprotein D, and the autophosphorylationof TrkB is detected using a labeled anti-phosphotyrosine antibody,thereby measuring the kinase activity of TrkB.

As described above, the present invention also provides a method ofscreening a therapeutic agent for ectopic pregnancy, the methodcomprising the following steps (a) to (d):

-   (a) preparing model animals in which human placental villi are    transplanted to a renal tissue of a mammal other than human;-   (b) administering a test sample to one (or one population) of the    model animals prepared and raising the animal(s), and administering    only the carrier in the test sample to another (or another    population) of the model animals prepared and raising the animal(s);-   (c) comparing cytotrophoblast cells and extravillous trophoblast    cells in the renal tissue in the model animal(s) to which the test    sample was administered with cytotrophoblast cells and extravillous    trophoblast cells in the renal tissue in the model animal(s) to    which the test sample was not administered; and-   (d) selecting the test sample as a therapeutic agent for ectopic    pregnancy, which test sample decreased cytotrophoblast cells and    extravillous trophoblast cells in the renal tissue in the model    animal(s) to which the test sample was administered.

Here, as the mammal other than human used in the above-described step(a) is preferably a rodent, and more preferably, a severeimmunodeficiency mouse which does not show rejection reaction againstthe placental villi originated from human. The duration of raising inthe above-described step (b) is preferably about 3 to 20 days in view ofcarrying out the screening quickly. The carrier in the above-describedstep (b) is the diluent such as a solvent, binder, vehicle, drugdelivery system or the like administered together with the test sample.Thus, a test in which the test substance and the carrier areadministered is carried out, and, as a control, a test in which thecarrier alone is administered is also carried out. For the comparisonbetween the cytotrophoblast cells and extravillous trophoblast cells inthe above-described step (c), it is preferred to use, although notrestricted thereto, cytokeratin which is a marker of trophoblast cellsfor the identification of the both trophoblast cells, and HLA-G which isa marker of extravillous trophoblast cells. Further, for the purpose ofdetecting cell growth, antibodies to proteins which can be employed asan index of cell growth, such as PCNA antibody and Ki-67 antibody mayalso be used.

Since a surgery is usually carried out when the oviduct is ruptured inectopic pregnancy, the ectopic pregnancy to be treated by thetherapeutic agent for ectopic pregnancy of the present invention isusually unruptured ectopic pregnancy

As will be concretely shown in the Examples below, the therapeutic agentfor ectopic pregnancy of the present invention suppresses the growth ofthe cytotrophoblast cells, thereby effectively treating ectopicpregnancy. Since the therapeutic agent for ectopic pregnancy is not ananticancer agent such as MTX, a systemic and severe side effect such asthat brought about by MTX is not resulted.

The present invention will now be described more concretely by way ofExamples. It should be noted that the present invention is notrestricted to the following Examples.

EXAMPLES Materials and Methods Human Villous Tissues

Human placental villi from first trimester (6-11 weeks), terminated forpsychosocial reasons, were obtained by dilatation and curettage, whereastissue samples of ectopic pregnancy were obtained from a patient at 8weeks of pregnancy by laparoscopic surgery in Akita University Hospital(Akita, Japan). Gestational age was determined by the date of the lastmenstrual period and ultrasound measurement of crown-rump length. Alltissue samples for in vitro and in vivo experiments were obtained fromJapanese women between 18 and 30 yr of age (mean, 23±4.5 yr) afterinformed consent in agreement with our regional medical ethicscommittee.

Human Trophoblast Villous Explant Culture

Preparation and cultivation of human villous explants of first trimesterplacentas were performed as described (Non-patent Document 13). Briefly,human placental villi at 6-8 weeks of gestation were asepticallydissected to remove decidual tissue and fetal membranes. Small pieces ofplacental villi (8 mg wet weight) were dissected under astereomicroscope (Leica Microsystems, Tokyo, Japan). Each villous piecewas put on Millicell-CM culture dish inserts (12-mm diameter)(Millipore, Bedford, Mass.) that were precoated with 200 μl of undilutedMatrigel Growth Factor Reduced (BD Bioscience pharmingen) and placed in24-well culture plates. The villous pieces were covered with 150 μl ofculture medium (DMEM/F12 without serum supplemented with 100 U/mlpenicillin, 100 μg/ml streptomycin, and 0.25 μg/ml ascorbic acid, pH7.4) (Invitrogen, Carlsbad, Calif.), whereas the bottom chambercontained 500 μl of culture medium. Villous pieces were cultured for 96h with or without different doses of the soluble ectodomain of TrkB (R&DSystems, Minneapolis, Minn.), a neurotrophic (pan)-specific Trk receptorinhibitor, K252a (Calbiochem, La Jolla, Calif.) (Non-patent Document14), or the inactive plasma membrane nonpermeable K252b (Calbiochem)(Non-patent Document 15) at 37° C. in 3% O₂/5% CO₂/92% N₂. Culture mediawere changed every 48 h and collected for measurement of glucoseconcentration.

Outgrowth of EVTs from the distal end of the villous tips (EVToutgrowth) and their migration into the surrounding Matrigel weremonitored daily using the stereomicroscopy, and cultures in which ≧50%anchoring villous tips showed cell outgrowth were classified as EVToutgrowth positive as described (Non-patent Document 13). At the end ofcultures, some villous explants including EVT outgrowth were subjectedto RNA extraction for real-time RT-PCR to quantify the transcript levelsof human leukocyte antigen-G (HLA-G). Glucose utilization by the villousexplants was calculated from the difference between glucoseconcentration in fresh medium and that in the conditioned medium after48 h of culture using an enzymatic assay (Mitsubishi BCL, Tokyo, Japan).The results were expressed as mg per 0.1 g wet weight tissue/48 h.

In some experiments, morphological changes in villous explants wereevaluated by hematoxylin and eosin (H&E) staining. Furthermore, theactivity of cellular proliferation was determined by immunohistochemicaldetection of proliferating cell nuclear antigen (PCNA) and Ki-67antigens. To measure the progression of apoptosis, some villous explantswere subjected to a quantitative caspase-3/7 enzyme assay as described(Non-patent Documents 12, 16). Apoptosis in villous explants was alsoassayed by detecting DNA fragmentation using in situ terminaldeoxynucleotidyl transferase-mediated dUDP nick end-labeling (TUNEL)(Non-patent Document 17).

Xenotransplantation of Human Villi into SCID Mice

To explore the roles of endogenous TrkB signaling in human ectopicpregnancy, xenotransplantation of human placental villi at 7-8 weeks ofgestation into SCID mice (C.B-17/Icr-scid/scidJcl) (CLEA Japan, Tokyo,Japan) was used as an in vivo model. The care and use of animals wasapproved by the Animal Research Committee, Akita University School ofMedicine. In preparation of grafts, small pieces of the placental villiwere dissected as described above and kept in ice-cold PBS untiltransplantation. After anesthetizing of the SCID mice at 8-11 weeks ofage with tribromoethanol (14-20 mg/kg) (Sigma, St. Louis, Mo.), the leftand right kidneys were sequentially exteriorized. A 0.5 mm incision wasthen made in each kidney capsule, and a piece of the placental villi (5mg wet weight) was transplanted underneath the capsule using a blunt tiptweezers. Treatment was initiated in animals after 1 week oftransplantation when murine vascular networks were known to appear inareas of cytotrophoblast invasion (Non-patent Document 18). Animalsweighted between 19-22 g on day of treatment. Intraperitoneal (ip)administration of K252a dissolved in physiological saline (500 μg/kg)was performed daily. For negative controls, treatment with K252b (500μg/kg) or vehicle alone was used. The doses of K252a and K252b chosenfor these experiments were based on previous studies (Non-patentDocuments 12, 19). Some animals were treated daily with methotrexate(ip; 1 mg/kg) (Sigma) corresponding to the therapeutic dose used formedical treatment of ectopic pregnancy (Non-patent Document 3). The micewere killed after 7 days of treatment. Kidneys with villi transplantswere excised and homogenized to measure hCG-β levels and caspase-3/7activities. To identify trophoblasts in the kidney, cytokeratin, amarker for trophoblasts (Non-patent Document 20), was detected byimmunohistochemistry. In addition to H&E staining, in vivo cellproliferation and apoptosis in the excised samples were evaluated byimmunostaining of PCNA and Ki-67, and the TUNEL assay, respectively.

Statistical Analysis

Chi-square test was performed to compare the proportion of EVT positivein villous explant cultures. One-way ANOVA, followed by Fisher'sprotected least significant difference test, was used to evaluate otherdifferences. Data are mean±SEM.

RT-PCR

For conventional RT-PCR to study the expression of neurotrophins (nervegrowth factor, NGF, and neurotroohin-3, NT-3) and Trk receptors (TrkA,and TrkC) in human placental villi, the primers for NGF, NT-3, TrkA,TrkC, and β-actin have been described (Non-patent Document 11). PCRreactions comprised 35 cycles of amplification with denaturation at 94°C. for 30 sec, annealing at 57° C. (TrkA), 60° C. (TrkC and β-actin), or62° C. (NGF and NT-3) for 30 sec, and elongation at 72° C. for 30 sec.For negative controls, no mRNA was included.

Real-Time RT-PCR

Quantitative real-time RT-PCR of TrkB and human leukocyte antigen-G(HLA-G) transcript levels in placental villi and mouse kidneys withxenotransplanted human villi was performed using a SmartCycler (Takara,Tokyo, Japan) with primers and hybridization probes for TrkB and β-actinas described (Non-patent Document 32). Primers for TrkB corresponded tothe catalytic kinase domain of the receptor to avoid the amplificationof truncated isoforms (Non-patent Document 33). Fragmented TrkB wasspecifically amplified using a reported primer (Non-patent Document 37).Validated Taqman gene expression assay was used to quantify theexpression of HLA-G (Applied Biosystems, Forster City, Calif.). Datawere normalized based on β-actin transcript levels.

Immunoassays

For ELISA, placental villi were homogenized in a buffer containing 137mM NaCl, 20 mM Tris-HCl, 1% Nonidet P40, 10% glycerol, and a proteaseinhibitor cocktail (Roche Applied Science, Indianapolis, Ind.) beforecentrifugation at 8,000×g for 5 min at 4 C. Quantification ofbrain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5)in placental villi was performed using ELISA as described (Non-patentDocument 32, Non-patent Document 10). The results were normalized byprotein concentrations and expressed as pg of BDNF or NT-4/5 per mg oftissues.

To localize BDNF and TrkB, BDNF and TrkB immunostaining in placentalvilli and villous tissues of ectopic pregnancy were performed asdescribed (Non-patent Document 11). BDNF antigen and TrkB antigen weredetected by rabbit anti-BDNF polyclonal antibody (Chemicon, Temecula,Calif.) and chicken anti-TrkB polyclonal antibody (Promega, Madison,Wis.) recognizing full length TrkB were used, respectively, afterdilution to 1:100. Some slides were subjected to proliferating cellnuclear antigen (PCNA) or Ki-67 immunostaining to evaluate cellularproliferation, whereas cytokeratin and HLA-G immunostaining wereperformed to identify trophoblasts and extravillous trophoblasts (EVTs)in the xenotransplanted human villi, respectively. Afterdeparaffinization and dehydration, antigen retrieval was performed byautoclave heating at 121° C. for 10 min (PCNA and Ki-67), treatment with0.4 mg/ml proteinase K (Sigma, St. Louis, Mo.) at room temperature for 5min (cytokeratin), or microwave heating in 10 mM citrate buffer (pH 6)for 3×3 min (HLA-G). Endogenous peroxidase activities were quenched with0.3% hydrogen peroxidase in methanol for 30 min. After blocking with 10%BSA-Tris-buffered saline (TBS) (Sigma) for 30 min, slides were incubatedwith either mouse anti-PCNA monoclonal antibodies (Cell SignalingTechnology, Danvers, Mass.), mouse anti-Ki-67 monoclonal antibodies(DAKO, Carpinteria, Calif.), rabbit anti-cytokeratin polyclonalantibodies (DAKO), or mouse anti-HLA-G monoclonal antibodies (Abeam,Cambridge, UK) at 1:4,000, 1:200, 1:1,000, or 1:500 dilution overnightat 4° C. After three washes in TBS, slides were incubated withbiotinylated anti-mouse or anti-rabbit secondary antibodies (Invitrogen,Carlsbad, Calif.) for 30 min at room temperature. After three washes,bound antibodies were visualized using a Histostain SP kit (Invitrogen).For negative controls, the primary antibody was replaced by nonimmunemouse IgG1 or nonimmune rabbit IgG (DAKO).

The human chorionic gonadotropin (hCG)-β protein levels in the renalhomogenates with transplanted villi were determined using RIA(Mitsubishi BCL, Tokyo, Japan) as described (Non-patent Document 12).

Results Expression of BDNF, NT-4/5, and TrkB in Human Placental VilliDuring Normal and Ectopic Pregnancy

Temporal expression of BDNF, NT-4/5, and TrkB in placental villi wasexamined by ELISA and real-time RT-PCR during first trimester of normalgestation. In the villi, ELISA analyses indicated that BDNF proteinlevels were 1.3- to 5.0-fold higher than those of NT-4/5 at all thepregnant days examined (FIG. 1A). BDNF protein levels were stable duringearly stage but decreased at 11 weeks of pregnancy, whereas NT-4/5protein levels were decreased after 7 weeks of pregnancy and maintainedat low levels during all pregnant stages examined (FIG. 1A). Incontrast, quantitative real-time RT-PCR analyses indicated that TrkBtranscript levels in placental villi were high at 6 weeks, and decreasedat 7 weeks of pregnancy, and gradually increased during pregnancyprogression (FIG. 1A).

Cell types expressing BDNF and TrkB proteins in normal placental villiwere determined by using immunohistochemistry. As shown in FIG. 1B,staining for BDNF and its receptor, TrkB were expressed in trophoblastcells of villi at 8 weeks of pregnancy in a cell type-specific manner.BDNF signal was detected in syncytiotrophoblasts and EVTs (FIG. 1B),whereas TrkB staining was localized to cytotrophoblasts and EVTs (FIG.1B). Similar cell type-specific expressions of BDNF and TrkB proteinswere detected in placental villi at 6-11 weeks of gestation (data notshown) and during ectopic pregnancy (FIG. 1C).

In vitro Inhibition of Endogenous TrkB Signaling Decreased HumanTrophoblast Growth

The expression of both TrkB ligands and receptors in specific cell typesof human placental villi suggests that the TrkB signaling system couldplay an autocrine/paracrine role during trophoblast growth. To determineif endogenous TrkB ligands act as a differentiation factor forcytotrophoblasts, we evaluated EVT outgrowth from cultured villousexplants treated with TrkB ectodomain and K252a. In controls, EVToutgrowth increased at 48 h of culture, and reached maximum levels at 96h of culture accompanied with shrinkage in explant sizes (FIG. 2A).Because a cell proliferation marker (Ki-67) was not found in themigrating cells (FIG. 7), the apparent outgrowth does not involve thedivision of villous cells. Treatment with either the TrkB ectodomain orK252a, but not the inactive K252b, suppressed EVT outgrowth in adose-dependent manner with similar efficacy (FIGS. 2A and B),accompanied with decreases in the transcript levels of HLA-G, a specificmarker for EVT (21) (FIG. 2C).

To examine effects of endogenous TrkB signaling on villous trophoblastproliferation, cellular functions were assessed morphologically and bymonitoring glucose metabolism. Consistent with the expression of TrkB inspecific cell types, treatment with either the TrkB ectodomain or K252a,but not the inactive K252b, decreased the number of villouscytotrophoblasts and induced partial detachment of trophoblast layersfrom villous stromal core at 96 h after culture (FIG. 3A, upper panels).Furthermore, we detected decreases in signals for two cell proliferationmarkers, PCNA (FIG. 3A, middle panels) and Ki-67 (FIG. 3A, lower panels)in remaining villous cytotrophoblasts following treatment with differentinhibitors. Non-proliferative syncytiotrophoblasts were not stained withboth markers in all of the control, TrkB ectodomain, K252a and K252bgroups tested. Treatment with the TrkB ectodomain and K252a alsodecreased the glucose utilization (>94% inhibition) by villous explants(FIG. 3B), confirming reduction of their cellular viability.

To determine if endogenous TrkB ligands act as survival factors forvillous trophoblasts, we evaluated apoptosis of cultured villousexplants treated with TrkB ectodomain and K252a. As shown in FIG. 4A,treatment with the TrkB ectodomain or K252a, but not the inactive K252b,increased the proportion of TUNEL-positive nuclei at 96 h after culture,thus suggesting the induction of apoptosis following suppression ofendogenous TrkB ligands. The increase of TUNEL-positive nuclei waspreferentially observed in cytotrophoblasts. In positive controlstreated with deoxyribonuclease I, all nuclei showed TUNEL signals,whereas no TUNEL-positive nuclei were observed in negative controls(data not shown). Furthermore, we detected increases in caspase-3/7activities by >6-fold in the villous explants following treatment withdifferent inhibitors (FIG. 4B).

Effects of a Trk Receptor Inhibitor on Trophoblast Growth in an in vivoAnimal Model of Ectopic Pregnancy

Our findings of the expression of TrkB ligands and receptors in humanvilli during both normal and ectopic pregnancies, and the in vitroinhibition of human trophoblast growth by TrkB inhibitors prompted us toinvestigate the effects of suppressing TrkB signaling as a potentialtreatment for ectopic pregnancy. Human placental villi werexenotransplanted into SCID mice as an in vivo model of ectopicpregnancy. Consistent with previous studies (Non-patent Document 18),trophoblast invasion into renal tissues was observed at 1 week afterxenotransplantation, and the invasion was extended to deeper regions ofkidney accompanied by increases in cell numbers three weeks later (FIG.5A). Increases in hCG-β levels in tissue homogenates (FIG. 5B) furthersuggested the transplanted villi developed at extrauterine site. Thetrophoblasts invaded into kidney were stained with HLA-G (FIG. 5C),indicating their differentiation into EVTs. These findings established amodel for ectopic pregnancy and allow us to test the use of Trkinhibitors for suppressing ectopic trophoblast growth.

We treated mice xenotransplanted with human villi with different drugsfor 7 days to evaluate their effectiveness to block ectopic pregnancy.Histopathological examination by cytokeratin, HLA-G, and H&E stainingand analysis of HLA-G-expressing transcript level by real-time RT-PCR inexcised kidney with transplanted villi showed decreases in cell numbersof invading EVTs and cytotrophoblasts in villous cores in the K252agroup (FIGS. 6A and B). There was also a major decreases in transcriptlevels for HLA-G (FIG. 6D), suggesting suppression of celldifferentiation and proliferation by K252a. PCNA (FIG. 6B, upper panel)and Ki-67 (FIG. 6B, middle panel) staining confirmed the effects ofK252a treatment on the suppression of cell proliferation. Decreases inhCG-β levels by 73.3% in tissue homogenates following K252a treatmentindicated a loss of cellular viability to synthesize hCG (FIG. 6E). Thiswas accompanied by increases in TUNEL-positive nuclei incytotrophoblasts after K252a treatment (FIG. 6C). We furthercharacterized apoptosis in transplanted villi by quantifying caspasesactivities and observed increased activation of caspase-3/7 by 4.1-foldwithin the xenografts of K252a-treated mice (FIG. 6F). Of importance,the inactive plasma membrane nonpermeable K252b was ineffective for allparameters tested. Furthermore, 1 mg/kg of MTX treatment did not inhibittrophoblast differentiation, proliferation, and survival (FIG. 6A-F).Similar to our previous studies (Non-patent Document 12), no obviousside effect was observed throughout experiments in all tested animals,and no body weight changes were found in K252a-treated group duringstudies (vehicle, 19.62±0.95 g; K252a, 19.27±1.03 g, and K252b,20.14±1.13 g).

1. A therapeutic agent for ectopic pregnancy, comprising as an effectiveingredient a suppressor of brain-derived neurotrophic factor (BDNF)and/or of brain-derived neurotrophic factor receptor (TrkB).
 2. Thetherapeutic agent according to claim 1, comprising as the effectiveingredient at least one selected from the group consisting of a tyrosinekinase inhibitor, a fragment thereof having an ability to bind to freeTrkB or BDNF, a modification thereof having a therapeutic effect forectopic pregnancy, a recombinant vector producing said fragment or saidmodification in a cell, an interfering RNA against BDNF gene or TrkBgene, a recombinant vector producing said interfering RNA in a cell, anantibody to BDNF or TrkB, an antisense nucleic acid against BDNF gene orTrkB gene and a recombinant vector producing said antisense nucleic acidin a cell.
 3. The therapeutic agent according to claim 2, comprising asthe effective ingredient at least one selected from the group consistingof a tyrosine kinase inhibitor, free TrkB and a TrkB fragment having anability to bind to BDNF.
 4. The therapeutic agent according to claim 3,wherein said tyrosine kinase inhibitor is a compound represented by thefollowing Formula (1):

(wherein a) both of Z¹ and Z² are hydrogen; 1) R is selected from thegroup consisting of OH, C₁-C₆ O-n-alkyl and C₂-C₆ O-acyl; 2) X isselected from the following group consisting of: H; CONHC₆H₅ with theproviso that in this case, R¹ and R² are not simultaneously Br; CH₂Ywherein Y is OR⁷ (wherein R⁷ is H or C₂-C₅ acyl); SOR⁸ wherein R⁸ isC₁-C₃ alkyl, aryl or a nitrogen-containing heterocyclic group; NR⁹R¹⁰wherein R⁹ and R¹⁰ are independently H or C₁-C₃ alkyl, Pro, Ser, Gly,Lys or C₂-C₅ acyl with the proviso that only one of R⁹ and R¹⁰ is Pro,Ser, Gly, Lys or acyl; SR¹⁶ wherein R¹⁶ is aryl, C₁-C₃ alkyl or anitrogen-containing heterocyclic group; N₃; CO₂CH₃; S-Glc; CONR¹¹R¹²wherein R¹¹ and R¹² are independently H, C₁-C₆ alkyl, C₆H₅ or C₁-C₆hydroxyalkyl, or R¹¹ and R¹² together form —CH₂CH₂OCH₂CH₂—; CH═NNHCONH₂;CONHOH; CH═NOH; CH═NNHC(═NH)NH₂;

CH═NN(R¹⁷)₂ wherein R¹⁷ is aryl; CH₂NHCONHR¹⁸ wherein R¹⁸ is lower alkylor aryl; or X and R together form —CH₂NHCO₂—, CH₂OH(CH₃)₂O—, ═O or—CH₂N(CH₃)CO₂; 3) R¹, R², R⁵ and R⁶ are independently H, or two or lessof these are F, Cl, Br, I, NO₂, CN, OH, NHCONHR¹³, CH₂OR¹³, C₁-C₃ alkyl,CH₂OCONHR¹⁴ or NHCO₂R¹⁴, wherein R¹⁴ is lower alkyl; CH(SC₆H₅)₂ orCH(—SCH₂CH₂S—); R¹ is CH₂S(O)_(p)R²¹ and R², R⁵ and R⁶ are H wherein pis 0 or 1, R²¹ is aryl, C₁-C₃ alkyl, a nitrogen-containing heterocyclicgroup,

or CH₂CH₂N(CH₃)₂; R¹ is CH═NHR²²R²³ and R², R⁵ and R⁶ are H, wherein R²²and R²³ are independently H, C₁-C₃ alkyl, C(═NH)NH₂ or anitrogen-containing heterocyclic group, or R²² and R²³ together form—(CH₂)₄—, —(CH₂CH₂OCH₂CH₂)— or —CH₂CH₂N(CH₃)CH₂CH₂—, with the provisothat R²² and R²³ cannot be simultaneously H, and that at least one ofR²² and R²³ is H except for the cases where both of these are alkyl; (b)in cases where Z¹ and Z² together represent O, X is CO₂CH₃, R is OH, andeach of R¹, R², R⁵ and R⁶ represents hydrogen).
 5. The therapeutic agentaccording to claim 4, wherein said tyrosine kinase inhibitor is K252a.6. The therapeutic agent according to claim 3, wherein the free TrkB orthe fragment thereof having an ability to bind to BDNF is one containingectodomain of TrkB which binds to BDNF.
 7. The therapeutic agentaccording to any one of claims 1 to 6, wherein said ectopic pregnancy isunruptured ectopic pregnancy.
 8. A method of screening a therapeuticagent for ectopic pregnancy, said method comprising measuring the kinaseactivity of TrkB in the presence of a test substance and the kinaseactivity of TrkB in the absence of said test substance; and selecting atest substance which decreases the kinase activity of TrkB.
 9. A methodof screening a therapeutic agent for ectopic pregnancy, said methodcomprising the following steps (a) to (d): (a) preparing model animalsin which human placental villi are transplanted to a renal tissue of amammal other than human; (b) administering a test sample to one (or onepopulation) of said model animals prepared and raising the animal(s),and administering only the carrier in said test sample to another (oranother population) of said model animals prepared and raisin theanimal(s); (c) comparing cytotrophoblast cells and extravilloustrophoblast cells in said renal tissue in said model animal(s) to whichsaid test sample was administered with cytotrophoblast cells andextravillous trophoblast cells in said renal tissue in said modelanimal(s) to which said test sample was not administered; and (d)selecting the test sample as a therapeutic agent for ectopic pregnancy,which test sample decreased cytotrophoblast cells and extravilloustrophoblast cells in said renal tissue in said model animal(s) to whichsaid test sample was administered.
 10. A suppressor of brain-derivedneurotrophic factor (BDNF) and/or of brain-derived neurotrophic factorreceptor (TrkB) for use in the treatment of ectopic pregnancy.
 11. Amethod of treating ectopic pregnancy, said method comprisingadministering an effective amount of a suppressor of brain-derivedneurotrophic factor (BDNF) and/or of brain-derived neurotrophic factorreceptor (TrkB) to a patient with ectopic pregnancy.